Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine the methylation status of female germ cells in reference to the programmed reversal of X chromosome inactivation in these cells, we examined human fetal ovaries at developmental stages from the time germ cells initiate meiosis to when they cease to synthesize DNA (8-21 weeks gestation). Using methylation-sensitive restriction enzymes, we analyzed 57 MspI sites (32 sites in the CpG islands, and 25 nonclustered sites) from five X-linked housekeeping genes (HPRT, G6PD, P3, PGK, and GLA) and two tissue specific genes (X-linked F9 and autosomal EPO). Methylation patterns were compared to those of male germ cells, sperm, and somatic tissues of both sexes. All 32 MspI sites in CpG islands were unmethylated in germ-cell fractions of fetal ovary and adult testes, which could explain the reversibility of X inactivation in these tissues. However, whereas male meiotic germ cells were extensively methylated outside the islands (in the body of genes) and the methylation patterns resembled those of most somatic tissues, none of the 25 nonclustered CpGs was methylated in DNA contributed by the germ-cell component of fetal ovaries. The presence of faint MspI-like fragments in HpaII digests of fetal testes as well as fetal ovary prior to the onset of meiosis suggests that DNA of primordial germ cells is unmethylated in both sexes. Our observations of meiotic germ cells suggest that the female germ cells remain unmethylated, but that methylation in male germ cells occurs postnatally, prior to or during the early stages of spermatogenesis. In any event, the striking sex difference in methylation status of endogenous single-copy genes in meiotic germ cells could provide a molecular basis for parental imprinting of the mammalian genome.
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PMID:Sex difference in methylation of single-copy genes in human meiotic germ cells: implications for X chromosome inactivation, parental imprinting, and origin of CpG mutations. 169 9

Alkylating agent damage was quantified in human T-lymphocytes by calculating gene-specific lesion frequencies and repair rates. At 3 time points after exposure to methyl methanesulfonate (0, 6, and 24 h), T-lymphocyte DNA was extracted, digested with HindIII, and divided into 2 aliquots. Apurinic sites were formed in the DNA fragments of both aliquots by heat-induced liberation of the N-methylpurines. The methoxyamine-treated aliquot provided gene fragments which were refractory to alkaline hydrolysis (full-length fragments), while the fragments in the untreated aliquot were cleaved at apurinic sites by hydroxide. After Southern blotting, lesion frequencies were calculated by comparing the band intensity of the full-length fragment to its unprotected counterpart. The restriction fragments analyzed were from the constitutively active dihydrofolate reductase (dhfr) plus hypoxanthine phosphoribosyltransferase (hprt) genes and from the transcriptionally inactive Duchenne muscular dystrophy gene (dmd). In decreasing order, the fragments containing the most lesions per kb of DNA were: hprt greater than dhfr greater than dmd. T-Lymphocytes from 2 females had 30% more heat-labile N-methylpurines in the active X-linked hprt gene than in the inactive X-linked dmd gene. The lesion frequency found in the male's lone hprt allele was the highest observed. These lesion frequency differences are discussed in terms of chromatin structure. After 6 and 24 h, no significant repair rate differences were observed among the 3 genes.
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PMID:Two expressed human genes sustain slightly more DNA damage after alkylating agent treatment than an inactive gene. 171 96

We recently reported a new X-linked mental retardation (XLMR) disorder in a four-generation family of Dutch descent. Features included Dandy-Walker malformation, basal ganglia disease, and seizures. Twenty-six family members, including two living affected males and two obligate carriers, were available for study. No evidence of linkage was observed between the disease locus and RFLPs from several X-chromosome regions, including Xp21-p22 (13 markers), proximal Xq (four markers), and Xq28 (three markers). However, a new hypervariable short tandem repeat (STR) within the HPRT gene at Xq26 showed positive linkage to the disease locus, with a maximum lod score of 2.19 at a recombination fraction of 0. A second hypervariable marker in Xq26, the dinucleotide repeat XL90A3 (DXS425), showed a lod score of .84 at a recombination fraction of .11. Both the HPRT and DXS425 markers were typed in 40 CEPH families, and subsequent multipoint linkage analysis showed the following order: Xcen-DXS425-(HPRT,XLMR)-F9-qter. HPRT and these flanking markers are therefore useful for carrier detection and prenatal diagnosis in this family. This study illustrates that hypervariable STRs will be powerful tools for linkage analysis and genetic diagnosis, particularly when relatively small families are involved.
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PMID:Linkage of the gene for an X-linked mental retardation disorder to a hypervariable (AGAT)n repeat motif within the human hypoxanthine phosphoribosyltransferase (HPRT) locus (Xq26). 174 58

Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frame-shifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. We have combined PCR and DGGE to: (i) Localize mutations in the X-linked human androgen receptor gene. PCR/DGGE was used to screen the individual exons in the 2757-bp coding region of the gene in afflicted individuals as well as in potential carriers. Inheritance of a mutant allele has been demonstrated in several cases; (ii) Analyze thousands of thioguanine-resistant mutants simultaneously. The in vitro mutational spectra of MNNG, ICR-191, and cisplatin at the human HPRT locus have been examined by this method. The compounds all have mutational hotspots in a GGGGGG sequence in exon 3; however, the particular mutations induced by the agents were different; (iii) Examine the fidelity of several DNA polymerases used in PCR. The fidelity of Thermus aquaticus DNA polymerase (Taq) is 1-2 x 10(-4) misincorporations/bp/replication. Problems with Taq polymerase arise in the analysis of complex mutant populations by DGGE because the Taq-induced errors reduce the sensitivity of the system. To circumvent this, it had been necessary to use Sequenase, a modified T7 DNA polymerase with a higher fidelity. However, Sequenase is not thermostable and must be added every PCR cycle. A thermostable DNA polymerase from Thermococcus litoralis (Vent) is now available, and we have examined the fidelity of Vent, Taq, and Sequenase polymerase in PCR using DGGE. The fidelity of Vent, Taq, and Sequenase polymerase was 2.4 x 10(-5), 8.9 x 10(-5), and 4.4 x 10(-5) errors/bp, respectively. Vent polymerase had the highest fidelity of the three enzymes tested.
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PMID:Analysis of mutations using PCR and denaturing gradient gel electrophoresis. 174 86

We used the X-linked restriction fragment length polymorphism (RFLP)-methylation strategy to study the clonal basis of the myelodysplastic syndrome (MDS) in seven patients. RFLP-methylation analysis was performed on cell populations from bone marrow (BM) aspirates and peripheral blood using probes specific for the hypoxanthine phosphoribosyltransferase (HPRT) or phosphoglycerate kinase (PGK) gene regions. Density gradient centrifugation methods were used to separate granulocytes and monocytes, and T lymphocytes were positively selected by CD2 (a pan-T marker) immunoconjugated magnetic beads. Cell populations from BM aspirates in 6 of the 7 patients with MDS showed a monoclonal pattern of X-inactivation. The neutrophilic and T-lymphocytic cell fractions were analyzed in 4 of the 6 patients, and the monocytic cell fraction in one of these, and all fractions analyzed showed a similar monoclonal pattern. In 2 of the latter 4 patients, both of whom had normal karyotypes, DNA from a skin biopsy showed a polyclonal pattern. Our data suggest that MDS is a clonal disorder, even in the absence of detectable cytogenetic abnormalities, and that the abnormal clone is capable of myeloid, monocytic, and lymphoid differentiation.
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PMID:Clonal studies in the myelodysplastic syndrome using X-linked restriction fragment length polymorphisms. 197 Apr 87

We have studied the mutagenicity and toxicity of physical and chemical agents in the Chinese hamster ovary (CHO) cell line K1-BH4 and its transformant, AS52. The AS52 cells lack the normal X-linked mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene but instead contain a single autosomally integrated copy of the bacterial equivalent, the xanthine-guanine phosphoribosyltransferase (gpt) gene. We found that X-rays and neutrons appear to be equitoxic to both cell types; however, these physical agents are approximately 10 times more mutagenic to the gpt gene of AS52 cells than to the hprt gene of K1-BH4 cells. We reasoned that if reactive oxygens were to mediate the mutagenic effects of both radiomimetic chemicals and radiation, then reactive oxygen-producing chemicals, such as streptonigrin and bleomycin, and oxidizing agents such as potassium superoxide and hydrogen peroxide, would exhibit similar levels of toxicity but different frequencies of mutants when assayed with the two cell lines. Our experiments fulfill such predictions. We postulate that the apparent hypermutability of AS52 cells probably results from a higher recovery of multi-locus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. Preliminary studies, using Southern blot and the polymerase chain reaction to analyze the mutational spectrum of the mutants, support our hypothesis that reactive oxygens induce deletion mutations in mammalian cells.
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PMID:Molecular analysis of reactive oxygen-species-induced mammalian gene mutation. 197 50

Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure.
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PMID:Identification of 17 independent mutations responsible for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency. 201 42

A mouse cDNA probe homologous to the human MCF2 transforming sequence has been identified and partially cloned, and is used here to localize the gene on the mouse X chromosome. The human gene has been physically mapped to within 60 kb of the gene for coagulation factor IX, within a large conserved linkage group between the mouse and human genomes which extends from HPRT to G6PD on the X chromosomes of both mammalian species. In situ hybridization of the mouse Mcf-2 probe onto mouse metaphase chromosomes indicates that this gene lies in the same region of the X chromosome as Cf-9, the mouse gene for coagulation factor IX. Moreover, segregation of species-specific genomic DNA polymorphisms for Mcf-2 and Cf-9 in a total of 203 individuals derived from two large interspecific mouse backcross populations (which are also segregating for 17 other X-linked molecular markers) demonstrates that the mouse genes are separated by only 0.5 +/- 0.5 cM. Despite this short distance we were able to order Mcf-2 and Cf-9 relative to one another and other genes in this region. The mouse gene order Hprt-Cf-9-Mcf-2-G6pd predicts a similar ordering of genes on the human X chromosome, a gene order which has only recently been demonstrated by physical mapping. Thus, the map location and linkage relationships of the Mcf-2 gene are similar in man and mouse, and this unique protooncogenic locus is part of a conserved linkage group on the mammalian X chromosome.
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PMID:Localization of the mouse Mcf-2 (Dbl) protooncogene within a conserved linkage group on the mouse X chromosome. 226 64

The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Steroid sulfatase gene in XX males. 230 2

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.
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PMID:X chromosome reactivation in mouse embryonal carcinoma cells. 242 74


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